独活寄生汤含药血清对退变软骨细胞细胞色素C及pro-Caspase-9、3的影响

Effects of medicated serum containing Duhuojisheng Decoction on the expression of cytochrome C, pro Caspase-9 and pro Caspase-3 in degenerative chondrocytes

背景:前期研究表明细胞凋亡是关节软骨细胞的中心性特征,对软骨破坏起着支配性的作用,是关节软骨退行性改变的病理因素之一。目的:观察独活寄生汤含药血清对大鼠退变软骨细胞释放细胞色素C、pro Caspase-9和pro Caspase-3的影响,探讨独活寄生汤治疗骨性关节炎的可能分子生物学机制。方法:建立体外退变软骨细胞模型,分别给予独活寄生汤含药血清或空白血清干预24,48 h,收集软骨细胞,Western Blot法检测干预后退变软骨细胞细胞色素C、pro Caspase-9和pro Caspase-3蛋白表达。结果与结论:随着干预时间的延长,在胞浆中空白血清组和含药血清组细胞色素C蛋白释放均逐渐降低,含药血清组释放较空白血清组明显减少(P<0.05);线粒体中空白血清组和含药血清组细胞色素C蛋白外漏均逐渐降低,含药血清组外漏较空白血清组明显减少(P<0.05)。空白血清组和含药血清组pro Caspase-9、pro Caspase-3蛋白量均逐渐升高,含药血清组升高更显著,且24 h组与48 h组之间差异有显著性意义(P<0.01)。结果提示独活寄生汤可以通过减少细胞色素C的释放,减少Caspase-9、Caspase-3的前体裂解,推测能有效抑制软骨细胞凋亡。

BACKGROUND： Previous studies have shown that apoptosis, a central feature of articular chondrocytes, plays a dominant role in cartilage damage, which is one of the pathological factors of articular cartilage degeneration. OBJECTIVE： To observe the effects of meditcated serum containing Duhuojisheng Decoction on the expression of cytochrome C, proCaspase-9 and proCaspase-3 in rat degenerative chondrocytes in vitro and to investigate the possible molecular biological mechanism of Duhuojisheng Decoction in the treatment of knee osteoarthritis. METHODS： A cultivation system of degenerative chondrocytes in vitro was established. After treatment with rneditcated serum containing Duhuojisheng Decoction or blank serum for 24 and 48 hours, the protein expression of cytochrome C, proCaspase-9 and proCaspase-3 was measured by western blot assay. RESULTS AND CONCLUSION： In the cytoplasm, the release of cytochrome C was reduced gradually in both groups in a time-dependent manner, and the release amount of cytochrome C was significantly lower in the medicated serum group than the blank serum group （P 〈 0.05）. In mitochondria, cytochrome C leakage was gradually decreased in both groups, and it was decreased significantly in the medicated serum group compared with the blank serum group （P 〈 0.05）. The protein expression of proCaspase-9 and proCaspase-3 was gradually increased in both groups, especially in the medicated serum group; the medicated serum containing Duhuojisheng Decoction could promote the protein expression of proCaspase-9 and proCaspase-3 in a time-dependent manner, and there was a significant difference at 24 and 48 hours （P 〈 0.01 ）. These findings indicate that the medicated serum containing Duhuojisheng Decoction can inhibit the apoptosis of osteoarthritis chondrocytes through inhibiting the release of cytochrome C and the activation of Caspase-9 and Caspase-3.